Repurposed antibiotics for non-nuclear genotoxic chemotherapy and pharmaceutical composition for anti-cancer containing the same

ABSTRACT

The present invention relates to a repurposed antibiotic compound for the treatment of cancer with minimal nuclear gene damage and an anticancer pharmaceutical composition comprising same. Since the repurposed antibiotic compound has a therapeutic effect in a manner that targets only the mitochondria of cancer cells, the modified antibiotic compound does not cause gene degeneration unlike conventional chemotherapy which damages nuclear DNAs to kill cancer cells, thereby preventing the recurrence of cancer. In addition, a mitochondria targeted therapy using the compound according to the present invention can effectively treat malignant tumors that are difficult to treat due to acquiring drug resistance by general anticancer treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a bypass continuation of PCT InternationalApplication No. PCT/KR2020/003248 filed Mar. 9, 2020, claiming prioritybased on Korean Patent to Application No. 10-2019-0030384 filed Mar. 18,2019, the respective disclosures of which are incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a modified antibiotic compound forcancer therapy with minimal damage to nuclear genes and an anticancerpharmaceutical composition including the same.

BACKGROUND ART

Numerous research groups around the world have made efforts to developvarious drug delivery systems for improving the therapeutic effects ofexisting anticancer drugs with reduced side effects, and as a result,succeeded in developing drug delivery systems that effectively deliverexisting highly toxic anticancer drugs to cancer tissues and allow theanticancer drugs to be activated only in the cancer microenvironment,achieving significantly improved safety in cancer treatment.

Despite these technological advances, however, the treatment andsuppression of recurrent and metastatic cancers with acquired drugresistance remain challenges for conventional anticancer therapies. 90%of cancer deaths are caused by recurrent cancers, not primary cancers.People who have had cancer have a very high probability of cancerrecurrence throughout their lives even after 5 years of survival.Especially, pediatric cancer patients have a more increased risk ofcancer recurrence. It has recently been reported that cancer recurrenceis paradoxically due to the use of anticancer drugs.

General chemotherapy inhibits the replication of nuclear DNA in cancercells and causes nuclear DNA damage to kill cancer cells. In thiscourse, mutations occur and will cause cancer recurrence. It was foundthat the administration of temozolomide to stage 2 glioma patients foranticancer therapy led to more malignant (stage 4) glioma as a recurrentcancer and DNA mutations induced by the anticancer drug temozolomidewere detected in the recurrent cancer.

Mitochondria are intracellular organelles that have their own circularDNA different from the nucleus in animals and divide and fuse in thesame way as prokaryotic cells. For these reasons, the origin ofmitochondria is explained by the endosymbiotic theory in which bacteriainvaded host cells during evolution and became mitochondria in animalcells. The evidence is that mitochondria have their own circular DNA,like bacteria, and are structurally and genetically similar to bacteria.Much research revealed that since mitochondria use enzymes forbiosynthesis, like bacteria, some antibiotics primarily targetingbacteria inhibit mitochondrial function to cause mitochondrial toxicity,which is responsible for their side effects. Attempts have been made tokill cancer cells by reversely using the toxicity of the antibiotics.However, the antibiotics do not exhibit significant toxicity to humancells at low concentrations and inhibit the division of both cancercells and normal cells (but even they are more selectively toxic tonormal cells) at high concentrations, making their use as anticancerdrugs substantially impossible. That is, the research results are simplylimited to the toxicity of the antibiotics to animal cells. Some studieshave also revealed that treatment of cancer stem cells with theantibiotics at high concentrations inhibits cancer growth. However, theantibiotics have no effect on bulk cancer cells, which account for 99%of the total cancer volume, and inhibit the division and growth ofcancer stem cells rather than kill cancer stem cells. That is, thesestudies simply suggest possible therapeutic effects of the antibiotics.

Cancer stem cells with high mitochondrial mass and high mitochondrialmembrane potential can be most effectively eliminated by drug deliverysystems that use the mitochondrial membrane potential to deliver andaccumulate drugs in mitochondria. Particularly, selective targeting ofmitochondria in cancer cells for effective cancer treatment wouldprevent cancer recurrence without causing genetic modification, unlikeconventional chemotherapy for killing cancer cells by nuclear DNAdamage.

DETAILED DESCRIPTION OF THE INVENTION Problems to be Solved by theInvention

The present invention intends to provide a modified antibioticanticancer drug that selectively kills cancer cells and cancer stemcells without damage to normal cells and normal stem cells, and afterthe lapse of a predetermined time, loses its targeting group to become asimple antibiotic drug that can be safely released from the body withoutsecondary toxicity, thus being useful for a novel cancer therapeuticstrategy to inhibit cancer recurrence and metastasis and minimize thepossibility of possible side effects.

Means for Solving the Problems

One aspect of the present invention provides a modified antibioticcompound for cancer therapy with minimal damage to nuclear genes,represented by Formula 1:

The structure and substituents of the antibiotic represented by Formula1 will be described below.

The modified antibiotic anticancer compound of the present inventionminimizes damage to nuclear genes in cancer cells and selectivelytargets mitochondria in cancer cells and thus it inhibits themitochondrial electron transport chain (ETC) and mitochondrial DNAsynthesis in cancer cells.

A further aspect of the present invention provides a pharmaceuticalcomposition for preventing and treating cancer diseases with minimaldamage to nuclear genes, including a modified antibiotic compoundrepresented by Formula 1 or a salt thereof as an active ingredient.

Effects of the Invention

The modified antibiotic anticancer drug of the present inventionselectively targets mitochondria in cancer cells to exhibit itstherapeutic effect. Thus, the modified antibiotic anticancer drug of thepresent invention can prevent cancer recurrence without causing geneticmodification, unlike conventional chemotherapy for killing cancer cellsby nuclear DNA damage. In addition, the compound of the presentinvention can be used to provide a mitochondria-targeted therapy foreffective treatment of malignant tumors that are difficult to treat bygeneral anticancer therapies due to their acquired drug resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the viabilities of metastatic breast cancer cells(MDA-MB-231) after treatment with a compound (Ester Mt-CFX) of thepresent invention and a commercial antibiotic (ciprofloxacin).

FIG. 2 shows the viabilities of metastatic breast cancer cells(MDA-MB-231) after treatment with Ester Mt-CFX and Amide Mt-CFX, whichwas obtained by replacing the binding site of Ester Mt-CFX with an amidegroup for better in vivo compatibility.

FIG. 3 shows the viabilities of various cancer cell types, includingA549 (lung cancer cells), SW620 (colon cancer cells), DU145 (prostatecancer cells), and PC3 (prostate cancer cells), after treatment with acompound (Ester Mt-CFX) of the present invention.

FIG. 4 shows the production of reactive oxidative species (ROS) in cellsby a compound (Ester Mt-CFX) of the present invention, which was labeledwith fluorescent markers (Amplex-red and CM-H2DCFDA).

FIG. 5 shows the inhibition of mitochondrial DNA synthesis and thereduction of membrane potential after treatment with a compound (EsterMt-CFX) of the present invention.

FIG. 6 shows the degrees of damage to DNA, protein, and lipid byreactive oxygen species generated after treatment of a compound (EsterMt-CFX) of the present invention.

FIG. 7 shows the reduction of mitochondrial membrane potentialdifferences (JC-1 assay) and the occurrence of apoptosis (Annexin Vassay) over time after treatment with a compound (Ester Mt-CFX) of thepresent invention.

FIG. 8 compares the degrees of damage to nuclear DNA and mitochondrialDNA by an anticancer compound (Ester Mt-CFX) of the present inventionand a commercial anticancer drug (doxorubicin (DOXO)).

FIG. 9 shows the expression levels of DNA repair proteins aftertreatment with a compound (Ester Mt-CFX) of the present invention and acommercial anticancer drug (doxorubicin (DOXO)) (where POLγ is a proteininvolved in mitochondrial gene repair and ERCC1 and DDB2 are proteinsinvolved in nuclear gene repair).

FIG. 10 compares the degrees of damage to mitochondrial genomic DNA byEster Mt-CFX and conventional anticancer drugs causing damage to nuclearDNA, which were determined by Taq1 assay.

FIG. 11 shows the results of real-time PCR for nuclear and mitochondrialDNA damage lesions after treatment with a compound (Ester Mt-CFX) of thepresent invention and conventional commercial anticancer drugs(doxorubicin, temozolomide, camptothecin, cisplatin, chlorambucil, and5-FU).

FIG. 12 shows the inhibitory effect of a compound (Ester Mt-CFX) ontumor cells using breast cancer transplantation models by determiningtumor-targeting effects over time (in vivo imaging) and measuringtime-dependent changes in tumor volume and weight after injection of thecompound.

FIG. 13 shows the viabilities of metastatic breast cancer cells(MDA-MB-231) after treatment with Ester Mt-CFX and Mt-CFX-4, which wasobtained by replacing the different positions of Mt(TPP).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in more detail.

The present invention is directed to a modified antibiotic drug for anew concept of anticancer therapy that can inhibit recurrence andmetastasis contributed from strong genotoxicity which is a major problemof current anticancer therapies.

The modified antibiotic anticancer drug compound of the presentinvention is represented by Formula 1:

wherein D is the backbone of a highly stable fluoroquinolone antibioticselected from flumequine, oxolinic acid, rosoxacin, cinoxacin, nalidixicacid, piromidic acid, pipemidic acid, ciprofloxacin, fleroxacin,lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin,rufloxacin, enoxacin, balofloxacin, grepafloxacin, levofloxacin,pazufloxacin sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin,gatifloxacin, moxifloxacin, sitafloxacin, prulifloxacin, besifloxacin,gemifloxacin, trovafloxacin, delafloxacin, danofloxacin, difloxacin,enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin, andsarafloxacin; X is connected to the terminal hydroxyl group of thebackbone of the fluoroquinolone antibiotic and is selected from O, S andNR (wherein R is selected from hydrogen, C₁-C₃₀ alkyl groups, C₆-C₃₀aryl groups, and C₂-C₃₀ heteroaryl groups), preferably O or NR; L is alinking group with the targeting group and is selected from C₁-C₃₀ alkylgroups, C₂-C₃₀ alkenyl groups, and polyalkylene glycol groups; Q is aGroup 15 element selected from N, P, As, and Sb; R₁ to R₃ are the sameas or different from each other and are each independently selected fromC₁-C₃₀ alkyl groups, C₂-C₃₀ alkenyl groups, C₆-C₃₀ aryl groups, andC₂-C₃₀ heteroaryl groups; and A⁻ is an anion selected from halogen,hydroxyl, carboxylate, sulfate, sulfamate, sulfonate, phosphate,phosphonate, boronate, and (poly)ethyleneoxy anions.

The modified antibiotic anticancer drug compound of the presentinvention uses the backbone of a highly stable antibiotic as a basicstructure and is designed to selectively target mitochondria, achievingenhanced anticancer therapeutic efficacy. In addition, the modifiedantibiotic anticancer drug compound of the present invention becomes asafe antibiotic drug after the mitochondria-targeting group ishydrolyzed in vivo, causing minimal side effects. When X is an amidegroup (NR), better in vivo stability of the compound represented byFormula 1 is ensured.

According to one exemplary embodiment of the present invention, thecompound of Formula 1 is selected from, but not limited to:

wherein n is an integer from 1 to 30 and A⁻ is as defined in Formula 1;and

wherein n and A⁻ are as defined above.

The compound of Formula 1 has the backbone of ciprofloxacin as afluoroquinolone antibiotic.

In the Examples section that follows, n is 6 and A⁻ is a chloride anion.Amide Mt-CFX is a compound obtained by replacing the binding site ofEster Mt-CFX with an amide group for better in vivo stability.

The present invention is also directed to a pharmaceutical compositionfor preventing and treating cancer diseases, including the modifiedantibiotic anticancer compound represented by Formula 1 or a saltthereof as an active ingredient.

The pharmaceutical composition of the present invention can be used toprevent and treat a wide range of cancer diseases such as primarycancers and their metastatic cancers. Examples of the cancer diseasesinclude, but are not limited to, breast cancer, lung cancer, coloncancer, prostate cancer, and metastatic cancers thereof.

The pharmaceutical composition of the present invention may be complexedwith other known drugs for the prevention or treatment of cancerdiseases before administration or may further include one or more otheradditives selected from carriers, diluents, adjuvants and stabilizers.

The dosage form of the composition according to the present inventionmay vary depending on the mode of administration desired. Examples ofsuch dosage forms include, but not limited to, solid, semi-solid, andliquid formulations such as tablets, pills, powders, capsules, gels,ointments, emulsions, and suspensions. The composition of the presentinvention may be administered in unit dosage forms suitable for singleadministration of precise dosages. The composition of the presentinvention may be administered orally or parenterally. For parenteraladministration, the composition of the present invention may beadministered intravenously, subcutaneously or intramuscularly.

Depending on the formulation desired, the composition may furtherinclude one or more pharmaceutically acceptable carriers, diluents,adjuvants, and stabilizers, which are defined as aqueous-based vehiclescommonly used to formulate pharmaceutical compositions for humanadministration.

The term “carrier” means a substance that facilitates the incorporationof a compound into cells or tissues. Examples of suitable carriersinclude, but not limited to, carbohydrate-based compounds, such aslactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, andcellulose, gum acacia, calcium phosphate, alginate, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,water, syrups, salt solutions, alcohols, gum Arabic, vegetable oils,such as corn oil, cotton seed oil, soybean oil, olive oil, and coconutoil, polyethylene glycol, methyl cellulose, methyl hydroxybenzoate,propyl hydroxybenzoate, talc, magnesium stearate, and mineral oils,which are commonly used to formulate pharmaceutical compositions. Theterm “diluent” is defined as a substance diluted in water that candissolve the compound of interest as well as stabilize the biologicallyactive form of the compound. Examples of suitable diluents includedistilled water, physiological saline, Ringer's solution, dextrosesolution, and Hank's solution. The stabilizers can be selected from thegroup consisting of proteins, carbohydrates, buffers, and mixturesthereof. The composition of the present invention may optionally furtherinclude one or more additives. Examples of such optional additivesinclude, but not limited to, lubricating agents, wetting agents,sweetening agents, flavoring agents, emulsifying agents, suspendingagents, and preservatives.

Such additional additives as carriers, diluents, adjuvants, andstabilizers may be used in amounts effective to acquire pharmaceuticallyacceptable formulations in view of the solubility, biological activity,and other characteristics of the active ingredient.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be more specifically explained with referenceto the following examples. It will be appreciated by those skilled inthe art that these examples are merely illustrative and the scope of thepresent invention is not limited thereto.

Synthesis Example 1: Synthesis of Ester Mt-CFX

Ester Mt-CFX was synthesized according to Scheme 1:

Compounds 1 and 2 shown in Scheme 1 were synthesized according tomethods known in the art.

(1) Synthesis of Compound 3

Compound 1 (1 g, 1.975 mmol) and Compound 2 (852 mg, 1.975 mmol) weredissolved in dimethylformamide as a solvent, and then potassiumcarbonate (819 mg, 5.925 mmol) was slowly added thereto. The mixture wasstirred at 50° C. for 12 h. The reaction mixture was evaporated underreduced pressure to remove the solvent. The residue was purified bycolumn chromatography and dissolved in 50 mL of a mixed solution ofmethanol and distilled water (1/9). NaBF₄ was added to the solution,followed by stirring for 1 h. The reaction mixture was extracted withdichloromethane and distilled water and dissolved in methanol. Afteraddition of Dowex® 1×8 chloride, the resulting mixture was stirred for 6h. The reaction mixture was filtered and evaporated to remove thesolvent.

(2) Synthesis of Ester Mt-CFX

Compound 3 was dissolved in 18 mL of a mixed solution of 1,4-dioxane anddichloromethane (4/1), and then 6 mL of 4 N HCl in dioxane was addeddropwise thereto at 0° C. The mixture was stirred at room temperaturefor 12 h. The solvents were removed by evaporation under reducedpressure, affording Ester Mt-CFX.

¹H NMR (CDCl₃, 500 MHz): δ 8.50 (s, 1H), 7.87-7.70 (m, 15H), 7.66 (d,J=13.1 Hz, 1H), 7.36 (d, J=7.1 Hz, 1H), 4.29-4.23 (m, 2H), 3.69-3.60 (m,2H), 3.59-3.53 (m, 1H), 3.50-3.42 (m, 4H), 3.30-3.20 (m, 4H), 1.78-1.65(m, 6H), 1.61-1.53 (m, 2H), 1.39 (d, J=5.9 Hz, 2H), 1.14-1.07 (m, 2H)ppm.

Synthesis Example 2: Synthesis of Amide Mt-CFX

Amide Mt-CFX was synthesized according to Scheme 2:

Compounds 1 and 2 shown in Scheme 2 were synthesized according tomethods known in the art.

(1) Synthesis of Compound 4

Compound 1 (1 g, 1.975 mmol) was dissolved in 35 mL of a solution of 7 NNH₃ in methanol. The solution was stirred at room temperature for 3days. Thereafter, the reaction solution was evaporated under reducedpressure to remove the solvent. The residue was purified by columnchromatography.

(2) Synthesis of Compound 5

Compound 2 (1 g, 2.318 mmol), EDC hydrochloride (667 mg, 3.477 mmol),and 1-hydroxybenzotriazole hydrate (470 mg, 3.477 mmol) were dissolvedin dimethylformamide as a solvent. The solution was stirred at roomtemperature for 30 min. To the reaction solution were added DMAP (425mg, 3.477 mmol) and Compound 4 (1.025 g, 2.318 mmol). The resultingmixture was stirred for 12 h. The reaction mixture was evaporated underreduced pressure to remove the solvent. The residue was purified bycolumn chromatography and dissolved in 50 mL of a mixed solution ofmethanol and distilled water (1/9). NaBF₄ was added to the solution,followed by stirring for 1 h. The reaction mixture was extracted withdichloromethane and distilled water and dissolved in methanol. Afteraddition of Dowex® 1×8 chloride, the resulting mixture was stirred for 6h. The reaction mixture was filtered and evaporated to remove thesolvent.

(3) Synthesis of Amide Mt-CFX

Compound 5 was dissolved in 18 mL of a mixed solution of 1,4-dioxane anddichloromethane (4/1), and then 6 mL of 4 N HCl in dioxane was addeddropwise thereto at 0° C. The mixture was stirred at room temperaturefor 12 h. The solvents were removed by evaporation under reducedpressure, affording Amide Mt-CFX.

¹H NMR (MeOD, 500 MHz): δ 8.88 (s, 1H), 7.95-7.74 (m, 17H), 7.69 (d,J=7.2 Hz, 1H) 3.85-3.79 (m, 1H), 3.69-3.64 (m, 4H), 3.54-3.49 (m, 4H),3.49-3.41 (m, 4H), 1.77-1.70 (m, 2H), 1.70-1.58 (m, 4H), 1.53-1.42 (m,4H) ppm.

Experimental Example

1-1 Animal Cell Culture

Human metastatic breast cancer cell line MDA-MB-231 (human breast cancercells), lung cancer cell line A549 (human lung carcinoma cells), humancolon cancer cell line SW620 (human colon carcinoma cells), humanprostate cancer cell lines DU145 and PC3 (human prostate cancer cells)were cultured in RPMI1640 media and modified Eagle's media (MEM). Allmedia were supplemented with 10% inactivated fetal bovine serum (FBS)and 1% penicillin-streptomycin. All cell lines were cultured at 37° C.and 5% carbon dioxide.

1-2. Cell Viability Analysis

MDA-MB-231, A549, SW620, DU145, and PC3 cells were seeded into 96-wellplates and cultured overnight for stabilization. After treatment of thestabilized cells with Ester MT-CFX, Amide MT-CFX, and ciprofloxacin atvarious concentrations from 0 to 100 μM for 48 h, the amounts of LDH inliving cells were measured to determine cell viabilities. Data are shownas the mean±SE of three independent experiments.

1-3. Reactive Oxidative Species (ROS) Measurement and Analysis

The activity of hydrogen peroxide (H₂O₂) produced in cells was evaluatedby detection with Amplex® Red reagent(10-acetyl-3,7-dihydroxyphenoxazine; Thermo Fisher scientific). Here,Amplex® Red reagent reacts with H₂O₂ in a stoichiometric ratio of 1:1 inthe presence of peroxidase in cells to produce resorufin as a redfluorescent product. The fluorescence of resorufin can be analyzed atexcitation and emission wavelengths of 571 nm and 581 nm, respectively.The MDA-MB-231 cell line was seeded into a 96-well plate and treatedwith 10 μM Ester MT-CFX for 48 h. Thereafter, the amount of hydrogenperoxide was measured. A reaction mixture of 50 μM Amplex® Red reagentand 0.1 U/mL HRP was prepared in Krebs-Ringer phosphate buffer (145 mMNaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mM CaCl₂, 1.22 mMMgSO₄, 5.5 mM glucose, pH 7.35). 100 μL of the reaction mixture wasadded to each plate well, followed by incubation at 37° C. for 10 min.The brightness of fluorescence was measured at 530-560 nm excitation and590 nm emission wavelengths using a microplate reader. The MDA-MB-231cell line was seeded into a confocal dish. Cells were treated with 10 μMEster MT-CFX for 48 h, followed by incubation with 10 μM CM-H2DCFDA dyefor 30 min. CM-H2DCFDA dye is a known marker for reactive oxygen speciesin cells. The fluorescence of reactive oxygen species produced in cellswas imaged by confocal laser scanning microscopy at 492-495 nmexcitation and 517-527 nm emission wavelengths.

1-4. DNA Oxidation Measurement

Intracellular DNA oxidation by oxidative stress was analyzed byenzyme-linked immunoassay using a commercially available 8-OHdG ELISAkit. After treatment of the MDA-MB-231 cells with 10 μM Ester MT-CFX and10 μM CFX for 48 h, the samples were incubated with primary antibodiesat 37° C. and secondary antibodies at 37° C. for 1 h. The incubatedsamples were allowed to stand at room temperature for 15 min for colordevelopment. The absorbance values of the resulting solutions weremeasured at 450 nm using a multiplate reader.

1-5. Protein Carbonylation Measurement

Protein carbonyl groups are known to be important physiological markersof oxidative stress and reactive oxygen species (ROS) are liable todamage intracellular proteins. Protein carbonylation was determined byELISA. After treatment of MDA-MB-231 cells with 10 μM Ester MT-CFX and10 μM CFX for 48 h, 10 μL of 1 μg/uL of protein lysate from each samplewas denatured with 10 μL of 10% (w/v) SDS and derivatized with 20 μL of20 mM 2,4-dinitrophenylhydrazine (DNPH) solution prepared in TPA. Afterincubation at room temperature for 10 min with vortexing every 2 min,the reaction product was neutralized with 20 μL of 2 M Tris-C₁. A 3 μLaliquot of DNP-derivatized sample was diluted with 0.25 mL of adsorptionbuffer (20 mM NaHCO₃, 150 mM NaCl, 0.25% SDS, pH 8.5), and 100 μL ofdiluted sample was loaded on to a 96-well plate. The plate was coveredwith aluminum foil and incubated overnight at 4° C. After incubation,the sample wells were rinsed 5 times with PBST (0.5% Tween 20) andincubated with 200 μL of blocking buffer (1% BSA in adsorption buffer)at 37° C. for 1 h. The sample wells were then incubated with 100 μL ofblocking buffer containing goat anti-DNP antibody at room temperaturefor 1 h. Following incubation, the sample wells were rinsed 5 times withPBST, and incubated with HRP-conjugated rabbit anti-goat IgG antibody atroom temperature for 1 h. After washing 5 times with PBST, standardwells were incubated with 100 μL of TMB substrate at room temperaturefor 2-3 min for color development. The reaction was stopped with 100 μLof 0.5 M H₂SO₄ and the absorbance was measured at 450 nm and 690 nm.After subtraction of background absorbance at 690 nm, the carbonylcontent in each sample was determined using a standard curve of oxidizedBSA standard.

1-6. Lipid Peroxidation Measurement

Lipid peroxidation is one of the markers for the mechanism of celldamage and can be confirmed by measuring malondialdehyde (MDA), aproduct of lipid peroxidation. After treatment of the MDA-MB-231 cellswith 10 μM Ester MT-CFX and 10 μM CFX for 48 h, 1 mg/mL of each samplewas placed in phosphate buffer and incubated in a thermostatic bath at37° C. for 6 h. 0.5 mL of 0.75% thiobarbituric acid was added to 10 μLof a mixed solution of butylated hydroxytoluene (BHT) quenched with 10%TCA. The mixture was heated at 95° C. for 20 min, cooled, andcentrifuged at 780×g for 10 min. The absorbance of the resultingsolution was measured at 532 nm using a multiplate reader.

1-7. FACS Measurement

Annexin-V positive apoptotic cells were analyzed by flow cytometry. Tothis end, the MDA-MB-231 cells were treated with 10 μM Ester MT-CFX fordifferent periods of time (0, 6, 12, and 24 h) and cultured forindicated periods of time. Cells were harvested, washed twice withice-cold PBS, placed in BD FACS tubes (10⁶/mL), and cultured with 10μg/mL annexin V in PBS containing 10% FBS at room temperature for 30min. Subsequently, cells were washed with ice-cold PBS and annexin Vfluorescence was measured in the FL-1 channel using a FACSCalibur flowcytometer (BD, USA).

1-8. Mitochondrial Membrane Potential Measurement

Mitochondrial membrane potential differences were measured. To this end,the MDA-MB-231 cells were treated with 10 μM Ester MT-CFX for differentperiods of time (0, 6, 12, and 24 h) and cultured for indicated periodsof time. Cells were harvested, washed once with ice-cold PBS, andcultured with 2 M5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl carbocyaninechloride (JC-1) at 37° C. for 20 min. The JC-1 stained cells werecentrifuged at 1,300 g for 3 min and washed twice with ice-cold PBS.Mitochondrial membrane potentials of JC-1 monomer (green channel) andaggregate (red channel) were measured in different wavelength bands.

1-9. Real-Time PCR Analysis

RNA was extracted from each sample using TRIzol solution. cDNAsynthesized from the isolated RNA or intracellular genomic DNA extractedusing a genomic DNA isolation kit was used for real-time polymerasechain reaction (PCR).

The real-time PCR data were normalized by the expression ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expressionlevels were expressed as fold changes over the control. The results(mean±SE) were obtained in triplicate and a p value <0.05 was taken tobe statistically significant.

1-10. Animal Preparation

Five-week-old immunodeficient male BALB/c-nu-nu mice (Orient bio, Korea)weighing between 18-20 g were used. The animals were bred and housed ina facility maintaining a humidity of 30-40% and a temperature of 22±1°C. on a 12-h light/dark cycle. All animal experiments were approved bythe Ethics Committee for Animal Studies at Korea University. Animalswere treated in accordance with the protocol approved by the EthicsCommittee.

1-11. In Vivo Mouse Xenograft Models

MDA-MB-231 cells (5×10⁶ cells) were injected subcutaneously into6-week-old immunodeficient BALB/c-nu-nu mice weighing between 18-20 g toestablish tumor-bearing mouse models. Ciprofloxacin and Ester MT-CFXwere injected intravenously into the established tumor-bearing mousemodels three times a week. The lengths of the long and short axes oftumors were measured weekly. 12 weeks after the first injection of eachcompound, the mice were sacrificed and tumor samples were collected forfurther analysis. The results are shown as the mean±SE of the tumors of4 mice in each group. Each asterisk (*) indicates a significantdifference to the corresponding control.

1-12. In Vivo Fluorescence Imaging

The tumor-targeting ability of Ester MT-CFX and the distribution ofEster MT-CFX in each organ were evaluated. Using an IVIS Lumina SeriesIII Preclinical imaging system (PerkinElmer CO., USA), in vivo spectralfluorescence images were obtained from 1 h to 48 h after injection of 2μmol/kg of Ester MT-CFX into tumor-bearing mice via the tail vein.Filters used to obtain the in vivo images were measured at an excitationwavelength of 560 nm. The fluorescence images were deconvoluted usingthe multi-excitation spectral analysis function.

1-13. Statistical Analysis

The mean and standard error (SE) of each group were calculated fromthree independent experiments done in triplicate. Statisticallysignificant differences between groups were evaluated by one-wayanalysis of variance (ANOVA) using SAS software (version 9.0, Cary).When the ANOVA showed a significant difference, comparisons of groupmeans were performed using Student's t-tests. A p value <0.05 was takento be statistically significant.

Experimental Results

(1) The commercially available antibiotic ciprofloxacin has little or noapoptotic effect on MDA-MB-231 metastatic breast cancer cells. Incontrast, the inventive compound Ester Mt-CFX having a targeting groupshowed a strong apoptotic effect on MDA-MB-231 cells with an IC₅₀ of ˜15μM (FIG. 1).

FIG. 1 shows the viabilities of MDA-MB-231 tumor cell line (Korean CellLine Bank). MDA-MB-231 is a triple-negative breast cancer cell line.Currently, there is no anticancer drug that can effectively treattriple-negative breast cancer. Cells were cultured in DMEM supplementedwith 10% FBS at 37° C. and 5% CO₂. Cytotoxicity was determined by LDHassay. Cells were cultured in a dish for ˜24 h such that they remainedstably adherent to the bottom of the dish, followed by treatment withthe antibiotic ciprofloxacin and the inventive Ester Mt-CFX for 48 h. Asa result of repeated experiments, the IC₅₀ of ciprofloxacin wasestimated to be 1.65 M and that of Ester Mt-CFX was 20-30 μM,demonstrating that the apoptotic activity of Ester Mt-CFX against thecancer cells was ˜55,000-fold higher than that of ciprofloxacin. Inconclusion, the inventive anticancer drug has an excellent anticancereffect on triple-negative breast cancer.

(2) For better in vivo stability, Amide Mt-CFX was synthesized byconverting the binding site (ester group) of Ester Mt-CFX to an amidegroup. The apoptotic effect of Amide Mt-CFX on cancer cells was verifiedto be similar to that of Ester Mt-CFX (FIG. 2).

The same experimental procedure as described for Ester Mt-CFX (FIG. 1)was repeated for the inventive Amide Mt-CFX. The results are shown inFIG. 2. The inventive Amide Mt-CFX also showed an enhanced apoptoticeffect on cancer cells.

(3) The inventive compounds Ester Mt-CFX and Amide Mt-CFX showedapoptotic effects on various cancer cell types, including A549 (lungcancer cells), SW620 (colon cancer cells), DU145 (prostate cancercells), and PC3 (prostate cancer cells) as well as MDA-MB-231 metastaticbreast cancer cells, demonstrating their wide applicability (FIG. 3).

To demonstrate the applicability of the inventive anticancer drugs tovarious cancer cell types, the same experimental procedure as describedfor MDA-MB-231 metastatic breast cancer cells (FIG. 1) was repeated forA549, DU145, SW620, and PC3 tumor cell lines (Korean Cell Line Bank).The results are shown in FIG. 3. The inventive anticancer drugs werefound to be effective against various cancer cell types. Particularly,the inventive anticancer drugs had IC₅₀ values of 23.77 μM and 27.05 μMagainst A549 and SW620, respectively.

(4) The anticancer effect of the inventive modified antibiotic Mt-CFXcan be explained by two mechanisms: (i) the accumulation of the compoundMt-CFX in mitochondria to inhibit the electron transport chain (ETC), asa result of which reactive oxygen species are produced (FIG. 4), and(ii) the inhibition of mitochondrial DNA synthesis (FIG. 5).

FIG. 6 shows the degrees of damage to DNA, protein, and lipid byreactive oxygen species generated after treatment of the inventivecompound Ester Mt-CFX. FIG. 7 shows the reduction of mitochondrialmembrane potential differences (JC-1 assay) and the occurrence ofapoptosis over time after treatment with the inventive compound EsterMt-CFX.

(5) FIG. 8 shows the non-toxicity of the inventive anticancer compoundEster Mt-CFX to nuclear genes by comparing the degrees of damage tonuclear DNA and mitochondrial DNA by the inventive anticancer compoundEster Mt-CFX and a commercial anticancer drug (doxorubicin (DOXO)). As aresult, doxorubicin showed strong toxicity to nuclear genes whereas theinventive anticancer compound Ester Mt-CFX showed no toxicity to nucleargenes and selective toxicity to mitochondrial genes.

FIG. 9 shows the expression levels of DNA repair proteins aftertreatment with the inventive compound Ester Mt-CFX and a commercialanticancer drug (doxorubicin (DOXO)) (where POLγ is a protein involvedin mitochondrial gene repair and ERCC1 and DDB2 are proteins involved innuclear gene repair). FIG. 10 reveals that unlike commercial anticancerdrugs, the inventive Ester Mt-CFX targeted mitochondria, which wasdetermined by Taq1 assay.

After treatment with the inventive Ester Mt-CFX and various anticancerdrugs, DNA damage lesions were analyzed by PCR. The results are shown inFIG. 11. The commercial anticancer drugs caused many mutations innuclear genes and Ester Mt-CFX induced mitochondrial mutations.

(6) The inventive fluorescent anticancer compound Ester Mt-CFX wasselectively accumulated only in cancer tissues, which was confirmed byan in vivo experiment. Treatment with the inventive anticancer compoundEster Mt-CFX caused a significant reduction in cancer volume compared totreatment with the control, demonstrating that the inventive anticancercompound Ester Mt-CFX has a superior therapeutic effect on cancer (FIG.12).

(7) FIG. 13 shows the significance of relative position between Mt andCFX. Referring to FIG. 13, Mt-CFX-4 where Mt is bound to CFX at adifferent position from Easter Mt-CFX forms zwitterion by acid-basereaction between carboxylic acid and piperazine under physiologicalconditions. Due to this reaction, cell permeability is significantlydecreases and cytotoxicity does not occur.

Contrary to the above result, Ester Mt-CFX according to the presentinvention protects the carboxylic acid moiety with an ester functionalgroup, so zwitterion does not form. Therefore cell influx into themitochondria of cancer cells occurs efficiently due to the positiveeffect of phosphonium ion, leading to the result that the two drugs showa difference in the apoptotic effect. This implies that it ispreferrable that the position of Mt bound to CFX is determined not toform zwitterion by acid-base reaction between carboxylic acid andpiperazine.

INDUSTRIAL APPLICABILITY

The modified antibiotic anticancer drug of the present inventionselectively targets mitochondria in cancer cells to exhibit itstherapeutic effect. Thus, the modified antibiotic anticancer drug canprevent cancer recurrence without causing genetic modification, unlikeconventional chemotherapy for killing cancer cells by nuclear DNAdamage. In addition, the modified antibiotic anticancer drug of thepresent invention can effectively treat malignant tumors that aredifficult to treat by general anticancer therapies due to their acquireddrug resistance.

1. A modified antibiotic anticancer compound represented by Formula 1:

wherein D is a fluoroquinolone antibiotic; X is connected to D and isselected from O, S and NR (wherein R is selected from hydrogen, C₁-C₃₀alkyl groups, C₆-C₃₀ aryl groups, and C₂-C₃₀ heteroaryl groups); L isselected from C₁-C₃₀ alkyl groups, C₂-C₃₀ alkenyl groups, andpolyalkylene glycol groups; Q is selected from N, P, As, and Sb; R₁ toR₃ are the same as or different from each other and are eachindependently selected from C₁-C₃₀ alkyl groups, C₂-C₃₀ alkenyl groups,C₆-C₃₀ aryl groups, and C₂-C₃₀ heteroaryl groups; and A⁻ is an anionselected from halogen, hydroxyl, carboxylate, sulfate, sulfamate,sulfonate, phosphate, phosphonate, boronate, and (poly)ethyleneoxyanions.
 2. The modified antibiotic anticancer compound according toclaim 1, wherein D in Formula 1 is a fluoroquinolone antibiotic selectedfrom flumequine, oxolinic acid, rosoxacin, cinoxacin, nalidixic acid,piromidic acid, pipemidic acid, ciprofloxacin, fleroxacin, lomefloxacin,nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, enoxacin,balofloxacin, grepafloxacin, levofloxacin, pazufloxacin sparfloxacin,temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, moxifloxacin,sitafloxacin, prulifloxacin, besifloxacin, gemifloxacin, trovafloxacin,delafloxacin, danofloxacin, difloxacin, enrofloxacin, ibafloxacin,marbofloxacin, orbifloxacin, and sarafloxacin.
 3. The modifiedantibiotic anticancer compound according to claim 1, wherein thecompound of Formula 1 is selected from:

wherein A⁻ is as defined in claim 1 and n is an integer from 1 to 30;and

wherein A⁻ and n are as defined above.
 4. The modified antibioticanticancer compound according to claim 1, wherein the compound ofFormula 1 selectively targets mitochondria in cancer cells.
 5. Themodified antibiotic anticancer compound according to claim 1, whereinthe compound of Formula 1 inhibits the mitochondrial electron transportchain (ETC) and mitochondrial DNA synthesis in cancer cells.
 6. Apharmaceutical composition for preventing and treating cancer diseases,comprising the modified antibiotic anticancer compound according toclaim 1 or a salt thereof as an active ingredient.
 7. The pharmaceuticalcomposition according to claim 6, wherein the pharmaceutical compositionselectively targets mitochondria in cancer cells.
 8. The pharmaceuticalcomposition according to claim 6, wherein the pharmaceutical compositioninhibits the mitochondrial electron transport chain (ETC) andmitochondrial DNA synthesis in cancer cells.
 9. The pharmaceuticalcomposition according to claim 6, wherein the cancer diseases areselected from breast cancer, lung cancer, colon cancer, prostate cancer,and metastatic cancers thereof.